Effect of feeding regimes on amyloid adhesin production and identification of amyloid adhesin producers in activated sludge

Context. Non-enzymatic structural proteins called amyloid adhesins are present in activated sludge.

Gap. We hypothesize that the (strong) binding forces within activated sludge microcolonies are due to adhesin-like interactions.

Aim. 

• Identify operating conditions that stimulate or repress adhesin expression.
• Investigate relation between amyloid quantity and bioflocculation performance.
• Identify amyloids and amyloid-producing microcolony formers.

Methods. Two sequencing batch reactors inoculated with sludge from municipal and industrial wastewater treatment plants were monitored during 183 days. The 6-hour reaction cycle consisted of an anoxic and aerobic phase. In a first experimental phase, bioflocculation-inducing feast-famine conditions were applied by feeding once every cycle. In a second and third phase, the carbon and nitrogen feeding 'stress' was relaxed: intermediate (every 72 minutes) or nearly continuous feeding (every 20 minutes) was applied. The amyloid quantity was assessed using Thioflavin T labeling. Sludge morphology, dewaterability, settleability and shear sensitivity were monitored. These results are being complemented by 16S rRNA bacterial community analysis and identification and quantification of structural proteins by proteomics. 

Findings. 

• Amyloid production can be increased by strong feast-famine conditions in activated sludge initially containing a negligible amount of amyloids and in sludge initially containing a higher amount of amyloids (Figure 1, Phase 1).
• While a decrease was expected, more continuous feeding resulted in an increase of amyloid expression (Figure 1, Phase 3). The link with nitrifier wash-out and, therefore, altered conditions for denitrifiers was investigated.
• Expression of amyloid adhesins is often localized in distinguishable microcolonies. The used monitoring tools quantify the overall floc strength rather than the strength of the microcolony building blocks, which might explain why we could not verify the link with bioflocculation performance. 

Utilization. Classifying adhesins and understanding their role in bioflocculation can further develop or understand targeted bioflocculation remediation approaches in wastewater treatment and can be exploited in industrial biotechnology.